Chronic opiate abuse impairs neuronal development and causes cognitive deficits in addicts. In addition to acting on inhibitory GABAergic synapses, our recent study revealed that mu-opioid receptors (MOR) could regulate the stability of the dendritic spines, which are mainly excitatory glutamatergic synapses. Since morphine tolerance and dependence have been reported to decrease in transgenic mice lacking AMPA receptor subunits, changes in the stability of such dendritic spines may contribute to opiate addiction. The overall objective of this proposed project is to clarify the intracellular signaling pathways that are involved in opioid regulation of dendritic spines. Based on our and other's previous studies, we proposed the following central hypothetical model: Activation of the postsynaptic opioid receptors in excitatory synapses inhibits the activities of protein kinases and Rho GTPases. Particularly, the inhibition of Rac 1, a Rho GTPase, causes the subsequent collapse of dendritic protrusions and spines by altering actin cytoskeleton. Combined genetic, living imaging and electrophysiological techniques will be used to test this model in cultured dissociated neurons. We will test this model by pursuing three specific aims. 1. We will further characterize the roles of MORs and their internalization in postsynaptic modulation of dendritic spines. Both receptor internalization and spine plasticity have been proposed to be important for drug addiction and tolerance development. The elucidation of the mechanistic links between these two critical cellular events may shed new light on our understanding of opiate addiction. We will also determine why some clinical relevant opiates, such as methadone, cause concentration-dependent biphasic changes in dendritic spines. The dosage-response studies of clinical relevant opiates may potentially reduce addictive liability of these opiates in the future. 2. We will identify and determine the Rho GTPase that mediates opioid modulation of dendritic spines. This aim will determine how morphine induces plasticity of spines by altering the actin cytoskeleton. 3. We will identify and determine the protein kinase(s) that mediates opioid modulation of dendritic spines. This aim will determine the signaling link between MOR and Rac1. By completing these specific aims, we will have determined three major signaling steps that mediate morphine-induced collapse of spines: the receptor that mediates morphine's effect;the protein kinase(s) that mediates the morphine response;and the actin-interacting proteins that contribute to morphine's effect. The determination of these signaling steps will provide a major framework of the signaling pathway that is responsible for opioid-induced changes in the excitatory synapses, and thus will provide new information about opiate abuse at a molecular and cellular level.